Estrogen receptor modulators and uses thereof

ABSTRACT

A method for preventing, diagnosing, or treating a condition mediated by an estrogen receptor by administering to a patient in need thereof an effective amount of a compound of formula I, II, or a combination thereof: wherein R1, R3, R4, and R5 are independently selected from H, OH, and OR a ; R2 is selected from H, OH, and (C═O)(C 1-7 )alkyl; R a  is (C 1-7 )alkyl or (C═O)(C 1-7 )alkyl; or a derivative of the compound selected from N-oxide derivatives, prodrug derivatives, protected derivatives, isomers, and mixtures of isomers of the compound; or a pharmaceutically acceptable salt or solvate of the compound or the derivative. Compounds of formula I and II and pharmaceuticals compositions thereof are also presented.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/818,748, which was filed on Jul. 6,2006, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grantTEN-13753 awarded by the United States Environmental Protection Agency.

BACKGROUND OF THE INVENTION

Estrogens regulate a variety of physiological processes in mammals,including reproduction, bone integrity, cellular homeostasis, andcardiovascular and central nervous systems. Estrogen (17β-estradiol, E2)belongs to a family of steroid hormones that act through solubleintracellular receptors by binding to high-affinity receptors on targetcells. Upon activation by estrogens, these receptors translocate to thenucleus, where they function as ligand-dependent transcription factors.Aside from a few notable exceptions, estrogenic ligands exhibit similaraffinities for the two receptor subtypes ERα and ERβ.

The canonical therapy for ER-positive breast cancers employs the partialantiestrogen tamoxifen. Tamoxifen has clearly demonstrated improvedprognosis in disease recurrence and overall survival in the managementof early-stage breast cancer. Furthermore, recent clinical studiessuggest that tamoxifen can be used prophylactically as a chemopreventiveagent for hormone-dependent breast cancer.

However, drawbacks emanating from long-term treatment with tamoxifeninclude acquired clinical resistance and an increased risk forendometrial cancer and related uterotropic effects. Recognition of thesefactors has stimulated the active pursuit of alternative selective ERmodulators (SERMs) that exert differential agonist and antagonisticeffects in various estrogen target tissues. Raloxifene, another SERM inclinical use, was developed to improve the drug safety profile byavoiding some of the undesirable estrogen agonist actions of otherSERMs.

In view of their enormous medical potential, there is a great need forfast, reliable tools to identify potential SERMs.

SUMMARY OF THE INVENTION

The present invention relates to methods for preventing, diagnosing, ortreating a condition mediated by an estrogen receptor by administeringto a patient in need thereof an effective amount of a compound offormula I, II, or a combination thereof:

wHerein R1, R3, R4, and R5 are independently selected from H, OH, andOR_(a);

R2 is selected from H, OH, and (C═O)(C₁₋₇)alkyl;

R_(a) is (C₁₋₇)alkyl or (C═O)(C₁₋₇)alkyl; or

a derivative of said compound selected from the group consisting ofN-oxide derivatives, prodrug derivatives, protected derivatives,isomers, and mixtures of isomers of said compound; or a pharmaceuticallyacceptable salt or solvate of said compound or said derivative.

Also provided are compounds of formula I or II:

wherein R1, R3, R4, and R5 are independently selected from H, OH, andOR_(a);

R2 is selected from H, OH, and (C═O)(C₁₋₇)alkyl;

R_(a) is (C₁₋₇)alkyl or (C═O)(C₁₋₇)alkyl; or

a derivative of the compound selected from N-oxide derivatives, prodrugderivatives, protected derivatives, isomers, and mixtures of isomers ofthe compound; or a pharmaceutically acceptable salt or solvate of thecompound or the derivative;

provided that R3 is OH or OR_(a) when R1 is OH and R2 is (C═O)CH₂CH₃;and

R4 is H or OR_(a) when R5 is H.

Pharmaceutical compositions, which include an effective amount of thecompound of formula I, II, or a combination thereof and apharmaceutically acceptable carrier are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of estrogenic activity assays on “hits”retrieved by in silico virtual screening of a chemical database;

FIG. 2 illustrates the results of ER competitive binding assays of thethree lead antagonists; and

FIG. 3 is a molecular modeling study of ER-ligand complex; (A) human ERLBD is represented by white ribbon lines and the ligands 4-OH Tamoxifen,raloxifene, Compound B, and Compound A are shown in capped stickrepresentation; (B)-(E) hydrogen bonds are represented as dashed linesbetween residues of the binding pocket of ER LBD and the followingligands: 17β-estradiol (B), 4-hydroxy tamoxifen (C), Compound A (D), andCompound B (E).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for preventing, diagnosing, ortreating a condition mediated by an estrogen receptor.

As used above, and throughout the description of the invention, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

“Patient” means a mammal including a human.

“Estrogen Receptor” as defined herein refers to any protein in thenuclear receptor gene family that binds estrogen, including, but notlimited to, any isoforms or deletion mutations having thecharacteristics just described. More particularly, the present inventionrelates to estrogen receptor(s) for human and non-human mammals (e.g.,animals of veterinary interest such as horses, cows, sheep, and pigs, aswell as household pets such as cats and dogs). Human estrogen receptorsincluded in the present invention include the α- and β-isoforms(referred to herein as “ERα” and “ERβ”) in addition to any additionalisoforms as recognized by those of skill in the art.

“Estrogen Receptor Modulator” is defined herein as a compound that canact as an estrogen receptor agonist or antagonist of estrogen receptor.

“Selective Estrogen Receptor Modulator” (or “SERM”) is a compound thatexhibits activity as an agonist or antagonist of an estrogen receptor(e.g., ERα or ERβ) in a tissue-dependent manner. Thus, as will beapparent to those of skill in the art, compounds of the invention thatfunction as SERMs can act as estrogen receptor agonists in some tissues(e.g., bone, brain, and/or heart) and as antagonists in other tissuetypes, such as the breast and/or uterine lining.

“Effective amount” means an amount of compound of the present inventioneffective for treating estrogen receptor mediated diseases orconditions, and thus producing the desired therapeutic effect.

“Treat” or “treatment” or “treating” mean to lessen, eliminate, inhibit,improve, alter, or prevent a disease or condition, for example byadministration of an estrogen receptor modulator or a SERM.

“Estrogen receptor mediated diseases or conditions” include anybiological or medical disorder in which estrogen receptor activity isimplicated or in which the inhibition of estrogen receptor potentiatesor retards signaling through a pathway that is characteristicallydefective in the disease to be treated. The condition or disorder mayeither be caused or characterized by abnormal estrogen receptoractivity. Representative estrogen receptor-mediated disorders include,for example, osteoporosis, atherosclerosis, estrogen-mediated cancers(e.g., breast and endometrial cancer), Turner's syndrome, benignprostate hyperplasia (i.e., prostate enlargement), prostate cancer,elevated cholesterol, restenosis, endometriosis, uterine fribroiddisease, skin and/or vagina atrophy, Alzheimer's disease, androgenicalopecia, pregnancy, and pre- and post-menopausal associated conditions(e.g. libido, dryness, skin integrity, youthfulness, hot flashes, andthe like). Successful treatment of a subject in accordance with theinvention may result in the prevention, inducement of a reduction in, oralleviation of symptoms in a subject afflicted with an estrogenreceptor-mediated medical or biological disorder. Thus, for example,treatment can result in a reduction in breast or endometrial tumorsand/or various clinical markers associated with such cancers. Likewise,treatment of Alzheimer's disease can result in a reduction in rate ofdisease progression, detected, for example, by measuring a reduction inthe rate of increase of dementia.

“Alkyl” means aliphatic hydrocarbon group which may be branched orstraight-chained having about 1 to about 10 carbon atoms. Preferredalkyl is “lower alkyl” having about 1 to about 3 carbon atoms; morepreferred is methyl. Branched means that one or more lower alkyl groupssuch as methyl, ethyl, or propyl are attached to a linear alkyl chain.

“Prodrug” means a form of the compound of formula I suitable foradministration to a patient without undue toxicity, irritation, allergicresponse, and the like, and effective for their intended use. A prodrugis transformed in vivo to yield the parent compound of the aboveformula, for example by hydrolysis in blood. A thorough discussion isprovided in T. Higuchi and V. Stella, Pro-drugs as Novel DeliverySystems, Vol. 14 of the A. C. S. Symposium Series, and in Edward B.Roche, et., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference.

“Solvate” means a physical association of a compound of this inventionwith one or more solvent molecules. This physical association involvesvarying degrees of ionic and covalent bonding, including hydrogenbonding. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Representative solvates includeethanolates, methanolates, and the like. “Hydrate” is a solvate whereinthe solvent molecule(s) is/are H₂O.

It will be appreciated by those skilled in the art that compounds usedin the invention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, tautomeric, orstereoisomeric form, or mixture thereof, of a compound of the invention,which possesses the useful properties described herein, it being wellknown in the art how to prepare optically active forms (for example, byresolution of the racemic form by recrystallization techniques, bysynthesis from optically-active starting materials, by chiral synthesis,or by chromatographic separation using a chiral stationary phase). It isalso well known in the art and, for example, as illustrated hereinbelowhow to determine estrogen receptor activity using the standard testsdescribed herein, or using other similar tests.

One embodiment of the present invention is a method for preventing,diagnosing, or treating a condition mediated by an estrogen receptor byadministering to a patient in need thereof an effective amount of acompound of formula I, II, or a combination thereof:

wherein R1, R3, and R4 are independently selected from H, OH, andOR_(a);

R2 is selected from H, OH, and (C═O)(C₁₋₇)alkyl;

R5 is H or OH;

R_(a) is (C₁₋₇)alkyl or (C═O)(C₁₋₇)alkyl or

a derivative of the compound selected from N-oxide derivatives, prodrugderivatives, protected derivatives, isomers, and mixtures of isomers ofthe compound; or a pharmaceutically acceptable salt or solvate of thecompound or the derivative.

Preferably, R_(a) is CH₃ or (C═O)CH₃.

Preferred configurations of formula I are set forth in Table 1:

TABLE 1 R1 R2 R3 OH (C═O)CH₂CH₃ H OH (C═O)CH₂CH₃ OH OH OH OH OH H H OH HOH H OH H H OH OH H H H H H OH OR_(a) H H OR_(a) H OH OR_(a) OH OHOR_(a) H OR_(a) H H OR_(a) OH H OR_(a)

The compound wherein R1 is OH, R2 is (C═O)CH₂CH₃, and R3 is H isreferred to herein as “Compound A” or “MS_(—)1105” (Bionet Research,Cornwall, England).

Preferred configurations of formula II are set forth in Table 2:

TABLE 2 R4 R5 OH H OH OH H OH OR_(a) H OR_(a) OH

The compound wherein R4 is OH and R5 is H is referred to herein as“Compound B” or “3257-4404” (ChemDiv, Inc., San Diego, Calif.).

In one embodiment, the estrogen mediated condition is selected fromosteoporosis, atheroschlerosis, estrogen-mediated cancers (e.g. breastcancer or endometrial cancer), Turner's syndrome, benign prostatehyperplasia, prostate cancer, elevated cholesterol, restenosis,endometriosis, uterine fribroid disease, skin and/or vagina atrophy,Alzheimer's disease, androgenic alopecia, pregnancy, and pre- andpost-menopausal associated conditions (e.g. libido, dryness, skinintegrity, youthfulness, hot flashes, and the like).

Yet another embodiment includes administering a compound of formula I orII to a patient in need thereof in a preventative or prophylacticamount. The need for a prophylactic administration according to themethods of the present invention is determined via the use of well knownrisk factors. The effective amount of an individual compound isdetermined, in the final analysis, by the physician in charge of thecase, but depends on factors such as the exact estrogen mediatedcondition or disease to be treated, the severity of the condition ordisease and other conditions or diseases from which the patient suffers,the chosen route of administration other drugs and treatments which thepatient may concomitantly require, and other factors in the physician'sjudgment.

Another embodiment includes an in vitro method for diagnosing a diseaseor condition in a patient as estrogen receptor-mediated by exposing asample (e.g. cancer cells) from the patient to a labeled compound offormula I or II under conditions such that the labeled compound can bindto an estrogen receptor present in the sample. Suitable labels for thecompound are readily determinable by one of skill in the art andinclude, for example, radio-labels, fluorescent labels, luminescentlabels, and the like. The amount of estrogen receptor-bound compoundpresent in the sample can be determined by detecting and quantifying thesignal from the bound label.

The compounds used in the methods of the present invention may beprepared by employing procedures known in the literature starting fromknown compounds or readily prepared intermediates. Compounds can also beobtained from commercial suppliers, for example, Asinex Corp.(Winston-Salem, N.C.), Bionet Research (Cornwall, England), Sigma (St.Louis, Mo.), Maybridge (Geel, Belgium), and ChemDiv, Inc. (San Diego,Calif.).

In practice, a composition containing a compound of formula I or II maybe administered in any variety of suitable forms, for example, byinhalation, topically, parenterally, rectally, or orally. More specificroutes of administration include intravenous, intramuscular,subcutaneous, intraocular, intrasynovial, colonical, peritoneal,transepithelial including transdermal, ophthalmic, sublingual, buccal,dermal, ocular, nasal inhalation via insufflation, and aerosol.

A composition containing a compound of formula I or II may be presentedin forms permitting administration by the most suitable route. Theinvention also relates to administering compositions containing acompound of formula I or II which is suitable for use as a medicament ina patient. These compositions may be prepared according to the customarymethods, using one or more pharmaceutically acceptable adjuvants orexcipients. The adjuvants comprise, inter alia, diluents, sterileaqueous media and the various non-toxic organic solvents. Thecompositions may be presented in the form of oral dosage forms, orinjectable solutions, or suspensions.

The choice of vehicle and the compound of formula I or II in the vehicleare generally determined in accordance with the solubility and chemicalproperties of the product, the particular mode of administration and theprovisions to be observed in pharmaceutical practice. When aqueoussuspensions are used they may contain emulsifying agents or agents whichfacilitate suspension. Diluents such as sucrose, ethanol, polyols suchas polyethylene glycol, propylene glycol and glycerol, and chloroform ormixtures thereof may also be used. In addition, the compound of formulaI or II may be incorporated into sustained-release preparations andformulations.

For parenteral administration, emulsions, suspensions or solutions ofthe compounds according to the invention in vegetable oil, for examplesesame oil, groundnut oil or olive oil, or aqueous-organic solutionssuch as water and propylene glycol, injectable organic esters such asethyl oleate, as well as sterile aqueous solutions of thepharmaceutically acceptable salts, are used. The injectable forms mustbe fluid to the extent that it can be easily syringed, and properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prolonged absorption of theinjectable compositions can be brought about by use of agents delayingabsorption, for example, aluminum monostearate and gelatin. Thesolutions of the salts of the products according to the invention areespecially useful for administration by intramuscular or subcutaneousinjection. Solutions of the compound of formula I or II as a free baseor pharmacologically acceptable salt can be prepared in water suitablymixed with a surfactant such as hydroxypropyl-cellulose. Dispersion canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. The aqueous solutions, also comprising solutions ofthe salts in pure distilled water, may be used for intravenousadministration with the proviso that their pH is suitably adjusted, thatthey are judiciously buffered and rendered isotonic with a sufficientquantity of glucose or sodium chloride and that they are sterilized byheating, irradiation, microfiltration, and/or by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating the compoundof formula I or II in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze dryingtechnique, which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

Topical administration, gels (water or alcohol based), creams orointments containing the compound of formula I or II may be used. Thecompound of formula I or II may be also incorporated in a gel or matrixbase for application in a patch, which would allow a controlled releaseof compound through transdermal barrier.

For administration by inhalation, the compound of formula I or II may bedissolved or suspended in a suitable carrier for use in a nebulizer or asuspension or solution aerosol, or may be absorbed or adsorbed onto asuitable solid carrier for use in a dry powder inhaler.

Compositions according to the invention may also be formulated in amanner which resists rapid clearance from the vascular (arterial orvenous) wall by convection and/or diffusion, thereby increasing theresidence time of the particles at the desired site of action. Aperiadventitial depot comprising a compound according to the inventionmay be used for sustained release. One such useful depot foradministering a compound according to the invention may be a copolymermatrix, such as ethylene-vinyl acetate, or a polyvinyl alcohol gelsurrounded by a Silastic shell. Alternatively, a compound according tothe invention may be delivered locally from a silicone polymer implantedin the adventitia.

An alternative approach for minimizing washout of a compound accordingto the invention during percutaneous, transvascular delivery comprisesthe use of nondiffusible, drug-eluting microparticles. Themicroparticles may be comprised of a variety of synthetic polymers, suchas polylactide for example, or natural substances, including proteins orpolysaccharides. Such microparticles enable strategic manipulation ofvariables including total dose of drug and kinetics of its release.Microparticles can be injected efficiently into the arterial or venouswall through a porous balloon catheter or a balloon over stent, and areretained in the vascular wall and the periadventitial tissue for atleast about two weeks. Formulations and methodologies for local,intravascular site-specific delivery of therapeutic agents are discussedin Reissen et al. (Am. C'oll. Cardial. 1994; 23: 1234-1244), the entirecontents of which are hereby incorporated by reference.

A composition according to the invention may also comprise a hydrogelwhich is prepared from any biocompatible or non-cytotoxic (homo orhetero) polymer, such as a hydrophilic polyacrylic acid polymer that canact as a drug absorbing sponge. Such polymers have been described, forexample, in application WO93/08845, the entire contents of which arehereby incorporated by reference. Certain of them, such as, inparticular, those obtained from ethylene and/or propylene oxide arecommercially available.

For the treatment of restenosis, the compounds of the invention areadministered directly to the blood vessel wall by means of anangioplasty balloon, which is coated with a hydrophilic film (forexample a hydrogel) which is saturated with the compound, or by means ofany other catheter containing an infusion chamber for the compound,which can thus be applied in a precise manner to the site to be treatedand allow-the compound to be liberated locally and efficiently at thelocation of the cells to be treated. This method of administrationadvantageously makes it possible for the compound to contact quickly thecells in need of treatment.

The treatment method of the invention preferably consists in introducinga compound according to the invention at the site to be treated. Forexample, a hydrogel containing composition can be deposited directlyonto the surface of the tissue to be treated, for example during asurgical intervention.

Advantageously, the hydrogel is introduced at the desired intravascularsite by coating a catheter, for example a balloon catheter, and deliveryto the vascular wall, preferably at the time of angioplasty. In aparticularly advantageous manner, the saturated hydrogel is introducedat the site to be treated by means of a balloon catheter. The balloonmay be chaperoned by a protective sheath as the catheter is advancedtoward the target vessel, in order to minimize drug washoff after thecatheter is introduced into the bloodstream.

Another embodiment of the invention provides for a compound according tothe invention to be administered by means of perfusion balloons. Theseperfusion balloons, which make it possible to maintain a blood flow andthus to decrease the risks of ischaemia of the myocardium, on inflationof the balloon, also enable the compound to be delivered locally atnormal pressure for a relatively long time, more than twenty minutes,which may be necessary for its optimal action. Alternatively, achanneled balloon catheter (“channeled balloon angioplasty catheter”,Mansfield Medical, Boston Scientific Corp., Watertown, Mass.) may beused. The latter consists of a conventional balloon covered with a layerof 24 perforated channels, which perfuse via an independent lumenthrough an additional infusion orifice.

Various types of balloon catheters, such as double balloon, porousballoon, microporous balloon, channel balloon, balloon over stent andhydrogel catheter, all of which may be used to practice the invention,are disclosed in Reissen et al. (1994), the entire contents of which arehereby incorporated by reference.

The use of a perfusion balloon catheter is especially advantageous, asit has the advantages of both keeping the balloon inflated for a longerperiod of time by retaining the properties of facilitated sliding and ofsite-specificity of the hydrogel are gained simultaneously.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising a compound of formula I or II and poloxamer, suchas Poloxamer 407, a non-toxic, biocompatible polyol, commerciallyavailable (BASF, Parsippany, N.J.).

A poloxamer impregnated with a compound of formula I or II may bedeposited directly on the surface of the tissue to be treated, forexample during a surgical intervention. Poloxamer possesses essentiallythe same advantages as hydrogel while having a lower viscosity.

The use of a channel balloon catheter with a poloxamer impregnated witha compound of formula I or II is especially advantageous. In this case,the advantages of both keeping the balloon inflated for a longer periodof time while retaining the properties of facilitated sliding, and ofsite specificity of the poloxamer, are gained simultaneously.

In another embodiment, the compound of formula I or II is administeredas a properly formulated coating on a stent device. Examples of stentdevices to which a SERM has been applied are disclosed in U.S. Pat. No.6,471,979, the contents of which are incorporated herein by reference.

In one aspect, the coating on a stent device is formed by applyingpolymeric material in which the compound of formula I or II isincorporated to at least one surface of the stent device.

Polymeric materials suitable for incorporating the compound of formula Ior II include polymers having relatively low processing temperaturessuch as polycaprolactone, poly (ethylene-co-vinyl acetate) or poly(vinyl acetate or silicone gum rubber and polymers having similarrelatively low processing temperatures. Other suitable polymers includenon-degradable polymers capable of carrying and delivering therapeuticdrugs such as latexes, urethanes, polysiloxanes,styrene-ethylene/butylene-styrene block copolymers (SEBS) andbiodegradable, bioabsorbable polymers capable of carrying and deliveringtherapeutic drugs, such as poly-DL-lactic acid (DL-PLA), andpoly-L-lactic acid (L-PLA), polyolthoesters, polyiminocarbonates,aliphatic polycarbonates, and polyphosphazenes.

A porosigen may also be incorporated in the drug-loaded polymer byadding the porosigen to the polymer along with the therapeutic drug toform a porous drug-loaded polymeric membrane.

“Porosigen” means as any moiety, such as microgranules of sodiumchloride, lactose, or sodium heparin, for example, which will dissolveor otherwise be degraded when immersed in body fluids to leave behind aporous network in the polymeric material. The pores left by suchporosigens can typically be as large as 10 microns. The pores formed byporosigens such as polyethylene glycol (PEG), polyethyleneoxide/polypropylene oxide (PEO/PPO) copolymers, for example, can also besmaller than one micron, although other similar materials which formphase separations from the continuous drug loaded polymeric matrix andcan later be leached out by body fluids can also be suitable for formingpores smaller than one micron. The polymeric material can be applied tothe stent while the therapeutic drug and porosigen material arecontained within the polymeric material, to allow the porosigen to bedissolved or degraded by body fluids when the stent is placed in a bloodvessel, or alternatively, the porosigen can be dissolved and removedfrom the polymeric material to form pores in the polymeric materialprior to placement of the polymeric material combined with the stentwithin a blood vessel.

If desired, a rate-controlling membrane can also be applied over thedrug loaded polymer, to limit the release rate of the compound of theinvention. The rate-controlling membrane can be added by applying acoating form a solution, or a lamination. The rate-controlling membraneapplied over the polymeric material can be formed to include a uniformdispersion of a porosigen in the rate-controlling membrane, and theporosigen in the rate-controlling membrane can be dissolved to leavepores in the rate-controlling membrane typically as large as 10 microns,or as small as 1 micron, for example, although the pores can also besmaller than 1 micron. The porosigen in the rate-controlling membranecan be, for example sodium chloride, lactose, sodium heparin,polyethylene glycol, polyethylene oxide/polypropylene oxide copolymers,or mixtures thereof.

In another aspect, the coating on the stent device can be formed byapplying the compound of formula I or II to at least one surface of thestent device to form a bioactive layer and then applying one or morecoats of porous polymeric material over the bioactive layer, such thatthe porous polymeric material has a thickness adequate to provide acontrolled release of the compound.

In one aspect, the porous polymeric material is composed of a polyamide,parylene or a parylene derivative applied by catalyst-free vapordesposition. “Parylene” refers to a polymer based on p-xylylene and madeby vapor phase polymerization as described in U.S. Pat. No. 5,824,049,incorporated herein by reference.

Alternatively, the porous polymeric material is applied by plasmadeposition. Representative polymers suitable for plasma depositioninclude poly (ethylene oxide), poly (ethylene glycol), poly (propyleneoxide), and polymers of methane, silicone, tetrafluoroethylenetetramethyldisiloxane, and the like.

Other suitable polymer systems include polymers derived fromphotopolymerizable monomers such as liquid monomers preferably having atleast two cross linkable C—C (Carbon to Carbon) double bonds, and beinga non-gaseous addition polymerizable ethylenically unsaturated compound,having a boiling point above 100° C., at atmospheric pressure, amolecular weight of about 100-1500 and being capable of forming highmolecular weight addition polymers readily. More preferably, the monomeris preferably an addition photopolymerizable polyethylenicallyunsaturated acrylic or methacrylic acid ester containing two or moreacrylate or methacrylate groups per molecule or mixtures thereof.

Representative examples of such multifunctional acrylates are ethyleneglycol diacrylate, ethylene glycol dimethacrylate, trimethylopropanetriacrylate, trimethylopropane trimethacrylate, pentaerythritoltetraacrylate, or pentaerythritol tetramethacrylate. 1 amides of (meth)acrylic acid, such as N-methylol methacrylamide butyl ether are alsosuitable, N-vinyl compounds such as N-vinyl pyrrolidone, vinyl esters ofaliphatic monocarboxylic acids such as vinyl oleate, vinyl ethers ofdiols such as butanediol-1,4-divinyl ether and allyl ether and allylester are also suitable. Also included are other monomers such as thereaction products of di- or polyepoxides such asbutanediol-1,4-diglycidyl ether or bisphenol A diglycidyl ether with(meth) acrylic acid. The characteristics of the photopolymerizableliquid dispersing medium can be modified for the specific purpose by asuitable selection of monomers or mixtures thereof.

Other useful polymer systems include a polymer that is biocompatible andminimizes irritation to the vessel wall when the stent is implanted. Thepolymer may be either a biostable or a bioabsorbable polymer dependingon the desired rate of release or the desired degree of polymerstability. Bioabsorbable polymers that could be used include poly(L-lactic acid), polycaprolactone, poly (lactide-co-glycolide), poly(hydroxybutyrate), poly (hydroxybutyrate-co-valerate), polydioxanone,polyorthoester, polyanhydride, poly (glycolic acid), poly (D, L-lacticacid), poly (glycolic acidcotrimethylene carbonate), polyphosphoester,polyphosphoester urethane, poly (amino acids), cyanoacrylates, poly(trimethylene carbonate), poly (iminocarbonate), copoly (ether-esters)(e.g., PEO/PLA), polyalkylene oxalates, polyphosphazenes andbiomolecules such as fibrin, fibrinogen, cellulose, starch, collagen andhyaluronic acid. Also, biostable polymers with a relatively low chronictissue response such as polyurethanes, silicones, and polyesters couldbe used and other polymers could also be used if they can be dissolvedand cured or polymerized on the stent such as polyolefins,polyisobutylene and ethylene-alphaolefine copolymers; acrylic polymersand copolymers, vinyl halide polymers and copolymers, such as polyvinylchloride; polyvinyl ethers, such as polyvinyl methyl ether;polyvinylidene halides, such as polyvinylidene fluoride andpolyvinylidene chloride; polyacrylonitrile, polyvinyl ketones, polyvinylaromatics, such as polystyrene, polyvinyl esters, such as polyvinylacetate; copolymers of vinyl monomers with each other and olefins, suchas ethylene-methyl methacrylate copolymers, acrylonitril-styrenecopolymers, ABS resins, and ethylene-vinyl acetate copolymers;polyamides, such as Nylon 66 and polycaprolactam; alkyl reins,polycarbonates; polyoxymethylenes; polyimides, polyethers; epoxy reins,polyurethanes; rayon; rayon-triacetate; cellulose, cellulose acetate,cellulose butyrate; cellulose acetate butyrate; cellophane, cellulosenitrate; cellulose propionate; cellulose ethers; and carboxymethylcellulose.

In addition to plasma deposition and vapor phase deposition, othertechniques for applying the various coatings on the stent surfaces maybe employed. For example, a polymer solution may be applied to the stentand the solvent allowed to evaporate, thereby leaving on the stentsurface a coating of the polymer and the therapeutic substance.Typically, the solution can be applied to the stent by either sprayingthe solution onto the stent or immersing the stent in the solution.

The compound of formula I or II may be used in the treatment ofrestenosis in combination with any anticoagulant, antiplatelet,antithrombotic or profibrinolytic agent. Often patients are concurrentlytreated prior, during and after interventional procedures with agents ofthese classes either in order to safely perform the interventionalprocedure or to prevent deleterious effects of thrombus formation. Someexamples of classes of agents known to be anticoagulant, antiplatelet,antithrombotic or profibrinolytic agents include any formulation ofheparin, low molecular weight heparins, pentasaccharides, fibrinogenreceptor antagonists, thrombin inhibitors, Factor Xa inhibitors, orFactor VIIa inhibitors.

The percentage of compound of formula I or II in the compositions usedin the present invention may be varied, it being necessary that itshould constitute a proportion such that a suitable dosage shall beobtained. Obviously, several unit dosage forms may be administered atabout the same time. A dose employed may be determined by a physician orqualified medical professional, and depends upon the desired therapeuticeffect, the route of administration and the duration of the treatment,and the condition of the patient. In the adult, the doses are generallyfrom about 0.001 to about 50, preferably about 0.001 to about 5, mg/kgbody weight per day by inhalation, from about 0.01 to about 100,preferably 0.1 to 70, more especially 0.5 to 10, mg/kg body weight perday by oral administration, and from about 0.001 to about 10, preferably0.01 to 10, mg/kg body weight per day by intravenous administration. Ineach particular case, the doses are determined in accordance with thefactors distinctive to the patient to be treated, such as age, weight,general state of health and other characteristics, which can influencethe efficacy of the compound of formula I or II.

The compound of formula I or II used in the invention may beadministered as frequently as necessary in order to obtain the desiredtherapeutic effect. Some patients may respond rapidly to a higher orlower dose and may find much weaker maintenance doses adequate. Forother patients, it may be necessary to have long-term treatments at therate of 1 to 4 doses per day, in accordance with the physiologicalrequirements of each particular patient. Generally, the compound offormula I or II may be administered 1 to 4 times per day. Of course, forother patients, it will be necessary to prescribe not more than one ortwo doses per day.

The following non-limiting examples set forth hereinbelow illustratecertain aspects of the invention.

EXAMPLES

Employing computational (“in silico”) screening of databases ofcommercially available chemicals, previously unrecognized estrogeniccompounds were identified. Biological assays indicated that two of thesecompounds:

are strong estrogen receptor (ER) antagonists. Subsequent computer-aidedmolecular modeling studies, in which these two compounds were “docked”inside the estrogen binding pocket of ER, elucidated their mode of ERbinding.

The ER antagonist 4-hydroxy (4-OH) tamoxifen and ER agonistdiethylstilbestrol (DES) were selected as queries to search a virtual(i.e., computer accessible) database of ˜200,000 commercially availablecompounds. Both molecules possess high binding affinity for the ERαcomparable in strength to the endogenous hormone 17β-estradiol, andtogether they represent archetypical examples of ER ligands.

Numerous “hits” were identified including, as expected, known estrogeniccompounds. After elimination of the hits already reported as estrogenic,nine compounds were selected for further experimental validation oftheir estrogenic activity.

To assay each of the selected hits from the search, a 25 μM sample wastested using the NR peptide ERα ELISA kit (Active Motif, Carlsbad,Calif.) according to manufacturer's instructions. Relative units oflight intensity for each test compound were normalized to the vehiclecontrol (DMSO) and then expressed in percentage of ER activation.Compounds exhibiting over 100% ER activation were identified asagonists, and below 100% as antagonists based on profiles of positive(17β-estradiol) and negative (tamoxifen) controls (FIG. 1). The datareported here represent results of three independent determinations induplicate. Error bars represent the standard deviation of the threedeterminations. All compounds were dissolved in DMSO as vehicle controlwith final concentration of less than 2.5% of DMSO.

Of the nine test compounds, several exhibited appreciable ER antagonistactivity as compared with vehicle control (100% ER activation) andtamoxifen (35.7%): mifepristone (51.7%), Compound A (53.1%), andCompound B (69%). Compounds 5386105 and 8R-0263 exhibited weakantagonist activity (82.1% and 77.9%). Compared with the strong agonistsDES (178.6%) and 17β-estradiol (148.5%), the test compound 4-hydroxyestradiol (196.3%) exceptional potency. We later discovered that thiscompound was previously shown as estrogenic, consistent with our insilico prediction. The remaining three test compounds (8R-0263,ASN_(—)3780064, and SP-00944) showed only marginal estrogenic activity.

Mifepristone (RU486), the biologically active agent in the so-called“morning-after pill”, has been reported as a progesterone (PR) andglucocorticoid receptor (GR) antagonist. Although mifepristone has beenshown to bind PR and GR directly, its ability to bind ER has not beenpreviously reported to our knowledge. This unexpected finding of strongER binding by mifepristone could be due to the presence of both ER andPR in the MCF-7 nuclear extract. The similarity of the ligand bindingdomains (LBDs) of ER and PR might preclude discrimination by theELISA-based assay. Subsequent competitive binding assays were conductedto explore this possibility.

A gel filtration displacement assay for estrogen receptor alpha (ERα)was employed to assess competitive binding by ER antagonists among thetest compounds in 10 μM. ER binding assays were conducted in duplicatein 50 mM Tris-HCl, pH7.5, 1 mM EDTA, 20% Glycerol and 1 mM DTT buffer.Radioligands that were used include [6, 7-³H] estradiol (specificactivity 44 Ci/mmol, Amersham Biosciences). Binding assays wereconducted on ice in a volume of 1 mL with 10 ng of purified full-lengthERα (Active Motif, Carlsbad, Calif.) and 25 nM 3H-estradiol in finalconcentration; 10 μM 17β-Estradiol and 4-OH tamoxifen (Sigma) were usedto define positive displacements as competing ligands respectively.

Following 1-hour incubation, assays were terminated by filtrationthrough Whatman GF/B filters. Filters were soaked in Ecoscint liquidscintillation mixture (National Diagnostics, Somerville, N.J.) andfilter-bound radioactivity was counted using a Beckman LS 1071 counter.Counts were normalized to the negative controls (no cold ligand) as thepercentage of bound radiolabeled-estradiol remaining after separatingbound radioligand from free radioligand. The negative controls weretaken as 100% bound radioligand remaining. The lower the percentage ofbound radiolabeled-estradiol implies a stronger binding affinity by thetest compound as definitive evidence of true ER ligands. Results arereported as the average values of three independent experiments induplicate. Error bars represent standard deviation of the threedeterminations (FIG. 2).

The >100% retention of radiolabeled-estradiol upon addition ofmifepristone indicated that mifepristone, a well-known ligand of the PRand GR, does not bind to ER directly. Instead, mifepristone exerts itsanti-estrogenic activity indirectly through an ER-independent pathway.Since MCF-7 nuclear extract was used as the source for ER, the ELISA kitcould have captured PR instead of ER when treated with mifepristonebecause of the similarity between the LBDs of ER and PR.

Two remaining antagonists (Compound A and Compound B) were confirmed astrue ligands of ERα. Both showed significant radioligand displacement ofERα with 65.9% and 89.8% bound radioligand remaining, respectively (FIG.2). The positive controls, 17β-estradiol or 4-OH tamoxifen, weredetermined to have 61.5% and 48.1% bound radiolabeled-estradiolremaining respectively (FIG. 2). The present results indicate that theER affinity is higher for Compound A than for Compound B. In fact,Compound A is comparable to the positive controls (known ligands withhigh affinity to ER) in terms of percentage of displaced radioligand.

Molecular modeling studies were conducted on Compound A and Compound Bafter docking them inside the binding pocket of the ER LBD. The primaryobjectives were to glean insights about their relative bindingaffinities and functional effects as antagonists. This was achieved bycomparing their binding poses with 17 β-estradiol and 4-OH tamoxifen asthey appear in complex with the ER LBD x-ray crystal structures (1GWRand 3ERT, respectively). Visual inspection of Compound A and Compound Bimmediately revealed that they adopt an extended conformation spanning˜12 Å that is characteristic of high-affinity ER ligands such as 17β-estradiol and 4-OH tamoxifen (FIG. 3).

The competitive binding assays yielded the following order in terms ofbinding affinity: 17 β-estradiol≧4-OH tamoxifen≧Compound A>Compound B.Examination of the hydrogen bonding interactions for these ER ligandsrevealed a consistent pattern (FIG. 3). Specifically, hydrogen bonds areevident with the sidechains of three residues (Glu353, Arg394, andHis524) for the strong agonist 17 β-estradiol and two residues (Glu353and Arg394) for the strong antagonist 4-OH tamoxifen. The Arg sidechain,which contains the protonated guanidinium group, can participate inmultiple stable hydrogen bonds concurrently. Compound A forms a hydrogenbond with the sidechain of Glu353, but the key interaction with Arg394is replaced by a weaker hydrogen bond with the backbone NH of Met388.Compound B forms a single hydrogen bond, with Glu353. In summary, theweaker ER binding affinities of Compound A and Compound B compared with4-OH tamoxifen may be explained in terms of the disparity in strength(for Compound A) and number (for Compound B) of their hydrogen bonds.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and script of the invention, and all such variations are intendedto be included within the scope of the following claims.

1. A method for preventing, diagnosing, or treating a condition mediatedby an estrogen receptor, said method comprising administering to apatient in need thereof an effective amount of a compound of formula I,II, or a combination thereof:

wherein R1, R3, R4, and R5 are independently selected from the groupconsisting of H, OH, and OR_(a); R2 is selected from the groupconsisting of H, OH, and (C═O)(C₁₋₇)alkyl; R_(a) is (C₁₋₇)alkyl or(C═O)(C₁₋₇)alkyl; or a derivative of said compound selected from thegroup consisting of N-oxide derivatives, prodrug derivatives, protectedderivatives, isomers, and mixtures of isomers of said compound; or apharmaceutically acceptable salt or solvate of said compound or saidderivative.
 2. The method of claim 1, wherein R1 is OH, R2 is(C═O)CH₂CH₃, and R3 is H.
 3. The method of claim 1, wherein R4 is OH andR5 is H.
 4. The method of claim 1, wherein said condition is selectedfrom the group consisting of osteoporosis, atheroschlerosis,estrogen-mediated cancers, Turner's syndrome, benign prostatehyperplasia, prostate cancer, elevated cholesterol, restenosis,endometriosis, uterine fribroid disease, skin and/or vagina atrophy,Alzheimer's disease, androgenic alopecia, pregnancy, and pre- andpost-menopausal associated conditions
 5. The method of claim 1, whereinsaid estrogen-mediated cancer is breast cancer or endometrial cancer. 6.A compound of formula I or II:

wherein R1, R3, R4, and R5 are independently selected from the groupconsisting of H, OH, and OR_(a); R2 is selected from the groupconsisting of H, OH, and (C═O)(C₁₋₇)alkyl; R_(a) is (C₁₋₇)alkyl or(C═O)(C₁₋₇)alkyl; or a derivative of said compound selected from thegroup consisting of N-oxide derivatives, prodrug derivatives, protectedderivatives, isomers, and mixtures of isomers of said compound; or apharmaceutically acceptable salt or solvate of said compound or saidderivative; provided that R3 is OH or OR_(a) when R1 is OH and R2 is(C═O)CH₂CH₃; and R4 is H or OR_(a) when R5 is H.
 7. A pharmaceuticalcomposition comprising an effective amount of the compound of claim 6and a pharmaceutically acceptable carrier.
 8. A method for diagnosing anestrogen receptor-mediated condition in a patient comprising (a)exposing a sample from the patient to a labeled compound of formula I orII under conditions such that the labeled compound can bind to anestrogen receptor present in the sample and (b) detecting a signal fromthe labeled compound bound to the estrogen receptor.